We have studied the regulation of the key regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase). We will focus on the regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum. We expect that our studies will yield fundamental insights into the role of degradation in regulating this enzyme and cholesterol homeostasis. More generally, we also anticipate that our work will describe a mechanism for degradation of membrane proteins of the endoplasmic reticulum. As a rationale for our studies, we consider the accelerated degradation of HMG-CoA reductase in response to regulatory molecules as a two-part process: 1) reductase itself as the target of regulatory molecules and the target for proteolysis; and 2) the proteolysis system responsible for reductase degradation. We also have a simple, undoubtedly too simple, working hypothesis that reductase binds cholesterol within the membrane domain, and undergoes some structural change which makes it more susceptible to a constitutive protease(s) within the endoplasmic reticulum. Our experiments are designed to confirm or reject parts or all of this hypothesis. In Specific Aim 1 we refine our mutagenesis studies in the regions of membrane spans-2 and 6 which preliminary data suggest are critical regions within the reductase membrane domain for a cholesterol response. We have significantly improved a novel method for demonstrating cholesterol binding to the membrane domain of reductase and will now try to correlate loss of sterol responsiveness of various mutants with loss of sterol binding. In Specific Aim 2 we analyze proteolytic fragments of reductase to determine a proteolytic cleavage site(s) within the membrane domain which is likely in the span-8 region. We will also extend our mutagenesis analysis of the span-8 region since we have shown that mutants in this region are resistant to proteolysis. In Specific Aim 3, we will isolate and characterize mutants in the degradation process of CHO cells using FACS-based detection of a chimeric protein, HMGFP, consisting of the membrane domain of reductase, HM, linked to Green Fluorescent Protein, GFP. Specific Aim 4 relies on a novel in vitro assay for reductase degradation and resolution/reconstitution to biochemically define the protease(s) necessary for reductase degradation in the endoplasmic reticulum.